Ab titers from mice dosed with the IL-12(Low)/F1-V DNA vaccine were equivalent to those induced with the single IL-12(Low)/F1-Ag or IL-12(Low)/V-Ag DNA vaccine. BALB/c mice (five mice/group) were first nasally dosed with DNA vaccines on weeks 0, 1, and 2 followed by nasal boosts with recombinant F1-Ag plus 2.5 μg CT on weeks 8 and 9. (A) Serum anti-F1-Ag and (B) anti-V-Ag Ab titers were determined by ELISAs at 6 and 12 weeks (wk) after primary immunization (p.i.). nd, not detected.

IL-12(Low)/F1-V DNA vaccine confers optimal protection against pneumonic plague. Single Ag DNA vaccines (A) were less effective than the IL-12(Low)/F1-V DNA vaccine (B). (A) Mice were dosed four times at weeks 0, 1, 2, and 12 with the IL-12(Low)/* DNA vaccines F1-Ag (n = 5), V-Ag (n = 5), or β-Gal (n = 5) and boosted on weeks 8, 9, and 12 with recombinant F1-Ag. An additional group received recombinant F1-Ag (n = 5) only on weeks 8, 9, and 12; a negative-control group received PBS only (n = 5). All mice were challenged 44 days after the last immunization. Mice dosed with IL-12(Low)/V-Ag plus F1-Ag protein boost showed the best efficacy at 60%. The fraction of mice that survived obtained from vaccinated mice were compared to PBS-dosed mice, and significance was determined as follows: *, P ≤ 0.001; **, P ≤ 0.005. (B) Similar immunization regimen using DNA vaccine plus recombinant F1-Ag in panel A was applied to mice dosed with IL-12(Low)/F1-V (n = 5) or IL-12(High)/F1-V (n = 7), and the additional controls were given F1-Ag only (n = 5) or PBS only (n = 5). The fraction of mice that survived of the vaccinated mice were compared to PBS-dosed mice, and significance was determined as follows: *, P ≤ 0.001; **, P = 0.006; ***, P = 0.02. The values for the two IL-12/F1-V vaccines were significantly different (P = 0.007) (#).

IgA (A) and IgG (B) AFC responses by mice nasally vaccinated with IL-12(Low)/F1-V DNA and IL-12(High)/F1-V DNA vaccines. Mice were nasally dosed, as described in the legend to Fig. 3, and lymphoid tissues were isolated on week 14. Total splenic, head and neck lymph node (HNLN), nasal mucosa-associated lymphoid tissues (NALT), nasal passage (NP), submaxillary gland (SMG), small intestinal lamina propria (iLP), and Peyer's patch (PP) mononuclear cells were isolated from each DNA vaccine group (five mice/group/experiment) and evaluated in a B-cell ELISPOT assay to assess F1-Ag- and V-Ag-specific (A) IgA and (B) IgG AFCs, as well as total (A) IgA and (B) IgG AFCs. Values are the means plus standard errors of the means (error bars) of AFC responses taken from two experiments. In some instances, significant differences between the high-dose and low-dose IL-12 vaccines in the different tissues were detected: #, P < 0.05; ##, P < 0.01.

Plasmid maps and expression determinations for the bicistronic IL-12(Low) and IL-12(High) DNA vaccines. (A) Circular plasmid maps for IL-12(Low)/F1-V (pGET146-mIL-12) DNA and IL-12(High)/F1-V (pBud-IL-12) DNA vaccines. hEF1-HTLV prom, hybrid elongation factor 1α-human T-cell leukemia virus promoter; EMCV IRES, encephalomyocarditis virus internal ribosome entry site; hCMV-IA prom, human cytomegalovirus IA promoter; Ori and ori, origin; BGH PA, bovine growth hormone and polyadenylation signal; PEF1-α, promoter of human elongation factor 1-α; Zeo-R, zeomycin resistance; SV40 PA, simian virus polyadenylation signal. (B and D) Expression of IL-12 was determined by IL-12-specific ELISAs using supernatants from 293A cells transfected with the IL-12(Low)/* (B) or IL-12(High)/* (D) DNA vaccine. Data are the means plus standard errors of the means (error bars) of two or three experiments and analyzed using the Mann-Whitney U-test. Values that were significantly different from vector alone are indicated by the brackets and asterisks as follows: *, P < 0.05; **, P < 0.01. (C and E) To detect F1-Ag or V-Ag expression by the single IL-12(Low)/* DNA vaccines (C) or the IL-12(Low)/F1-V DNA and IL-12(High)/F1-V DNA vaccines (E), cell lysates from transfected 293A cells were subjected to Western immunoblot analysis using rabbit anti-F1-Ag or rabbit anti-V-Ag sera. Expression of F1-Ag could not be detected for any of the vaccines, but expression of V-Ag was detected. F1-V expression by both IL-12 DNA vaccines was equivalent. The positions of molecular mass markers (M) (in kilodaltons) are indicated to the left of the immunoblots.

Increased expression of IL-12 has no effect upon serum IgG or mucosal IgA Ab titers. BALB/c mice (10 mice/group) were nasally immunized, as described in the legend to Fig. 2, plus a final immunization was given on week 12 with the DNA vaccine and F1-Ag plus CT. A kinetics analysis was performed on the IL-12(Low)/F1-V DNA and IL-12(High)/F1-V DNA vaccines. (A) Serum IgG and fecal IgA Ab titers to F1- and V-Ags were monitored for 14 weeks. The values are shown as means ± standard deviations (error bars). (B) Nasal washes were performed at week 14, and mucosal IgA Ab titers were measured. The values are shown as means plus standard deviations (error bars).

IgG subclass responses to F1- and V-Ag in sera from IL-12(Low)/F1-V DNA- and IL-12(High)/F1-V DNA-vaccinated mice. The mouse sera evaluated were shown in Fig. 3. The endpoint titers are depicted as the means plus standard deviations (error bars) for 10 mice/group. IgG1 and other IgG subclass endpoint titers that were significantly different are indicated by the brackets and asterisks as follows: *, P < 0.05; **, P < 0.01.

Cytokine responses by mice primed with IL-12(Low)/F1-V DNA and IL-12(High)/F1-V DNA vaccines. BALB/c mice were nasally dosed with the DNA vaccines on weeks 0, 1, and 2, and on week 7, total lymphocytes were isolated from spleens, HNLNs, and PPs. Lymphocytes were Ag pulsed for 2 days, and IFN-γ, IL-4, IL-5, IL-10, and TGF-β CFC responses (A) and secreted IFN-γ, IL-6, IL-10, and IL-17 (B) were measured by cytokine-specific ELISPOT assay and sandwich ELISA, respectively. Values are shown as means plus standard errors of the means (error bars) for two experiments (16 mice/group). In some instances, significant differences (P < 0.05) in the different tissues in the two IL-12 DNA vaccine groups (#) or versus medium (Med) (*) were detected.